Novel strategy for rapid functional in vivo validation of oncogenic drivers in haematological malignancies

Tim Pieters,Frederique Van Rockeghem,Jinke D’Hont,Steven Goossens,Filip Matthijssens,André Almeida,Pieter Van Vlierberghe,Lieven Haenebalcke,Tino Hochepied,Sara T’Sas,Tim Lammens,Lindy Reunes,Beatrice Lintermans,Lisa Demoen,Kelly Lemeire,Geert Berx,Jody J Haigh

doi:10.1038/s41598-019-46853-x

Abstract

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1+ murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel anti-leukemic drugs in vivo.

Highlights

Next-generation sequencing has rapidly expanded our knowledge on the mutational landscape of human cancer
We further refined this methodology by including an eGFP-Firefly Luciferase reporter cassette together with the transgene of interest, in order to provide a robust and rapid pipeline for the generation of in vivo cancer models that can be readily used for preclinical drug evaluation studies
The pRMCE-DV3 destination vector contains a DNA fragment that is flanked by heterospecific Frt sites and is composed of the Control of cell death b (Ccdb) suicide gene that is surrounded by attR recombination sites, a PKG promoter and a translation initiation codon (ATG) for Neomycin Resistance (NeoR) gene reactivation

Summary

Introduction

Next-generation sequencing has rapidly expanded our knowledge on the mutational landscape of human cancer. Conditional gain of function transgenic mouse models have been extensively used to validate the oncogenic properties of putative driver genes of interest in a cell/tissue-specific manner Of note, these in vivo model systems allow the study of bona fide oncogenes in the context of their tumour micro-environment and in the presence of a functional immune system. As a proof-of-principle, we here generated four novel R26 KI mice that enable conditional overexpression of 3 putative oncogenes (Jarid[2], Runx[2], MN1) and one dominant negative allele of a tumour suppressor (dnETV6), which all have been previously associated with the pathobiology of human leukemia[7,8,9,10,11,12] Using these novel model systems, we show that overexpression of MN1 in the hematopoietic lineages results in spontaneous development of myeloid leukemia in vivo. We confirm that luciferase-positive primary murine AML and T-ALL/T-LBL cells are transplantable in secondary recipients and can be readily used for preclinical drug testing purposes

Methods

Results

Conclusion